Wen Jian-Jun, Garg Nisha Jain
Departments of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-1070, USA.
J Bioenerg Biomembr. 2008 Dec;40(6):587-98. doi: 10.1007/s10863-008-9184-4. Epub 2008 Nov 14.
In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O(2) (-)) and amplex red (detects H(2)O(2)), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the Q(Nf)-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Q(o) center that was different from the Q(i) center of O(2) (-) generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Q(o) site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-alpha-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Q(o) site of complex III resulted in increased electron leakage and O(2) (*-) formation in infected myocardium, and was controlled by PBN.
在本研究中，我们已明确了克氏锥虫感染期间心肌中活性氧（ROS）生成增强的细胞来源及机制。与正常对照组相比，感染小鼠的心脏线粒体中，二氢乙锭（检测超氧阴离子O₂⁻*）和安吖啶红（检测过氧化氢H₂O₂）的ROS特异性荧光分别增加了63.3%和30.8%。感染小鼠心脏线粒体中ROS水平的升高分别与谷氨酸/苹果酸（复合体I底物）和琥珀酸（复合体II底物）支持的ROS释放速率增加59%和114%相关，与正常对照组相比，呼吸链电子泄漏速率最高增加74.9%。对正常心脏线粒体的抑制研究表明，鱼藤酮在复合体I的Q(Nf)-泛半醌位点诱导ROS生成。在复合体III中，黏噻唑从位于Q(o)中心的一个位点诱导ROS生成，该位点与抗霉素产生O₂⁻*的Q(i)中心不同。在感染小鼠的心脏线粒体中，正向（从复合体I到复合体III）和反向（从复合体II到复合体I）电子传递过程中复合体I的电子泄漏速率并未增强，复合体I不是感染心肌中ROS产生增加的主要位点。相反，靠近Q(o)位点的复合体III缺陷导致感染小鼠心脏线粒体中电子泄漏增强和ROS形成。用苯基-α-叔丁基硝基酮（PBN）治疗感染小鼠可改善呼吸链功能，随后降低电子泄漏和ROS释放程度。总之，我们表明复合体III的Q(o)位点受损导致感染心肌中电子泄漏增加和O₂⁻*形成，并受PBN调控。
